1. Field of the Invention
This invention relates to industrial scale preservation of sensitive biological materials for storage at temperatures above 0xc2x0 C. More particularly, the invention relates to technological processes and equipment for effecting the industrial scale dehydration of solutions and suspensions by foam formation, with optional stability drying to achieve the glass state, and optional milling to form dry powders.
2. Description of the Related Art
The preservation and storage of solutions or suspensions of biologically active materials, viruses, cells and small multicellular specimens is important for food and microbiological industries, agriculture, medical and research purposes. Storage of these dehydrated biologically active materials carries enormous benefits, such as reduced weight and reduced storage space, and increased stability.
Suggestions in the prior art for providing preservation of sensitive biological materials in dehydrated form include freeze-drying and vacuum or air-desiccation. Both, freeze-drying and desiccation preservation methods have positive and negative characteristics. While freeze-drying methods are scaleable to industrial quantities, conventional vacuum and air-desiccation methods do not yield preparations of biological materials which are scalable to industrial quantities. Freezing and other steps of the freeze-dying process are very damaging to many sensitive biological materials. The freeze-drying process is very long, cost ineffective, and cannot be performed using barrier technology to insure sterility of the material.
Some of the problems associated with preservation by freezing and drying have been addressed by addition of protectant molecules, especially carbohydrates, which have been found to stabilize biological materials against the stresses of freezing and drying. However, despite the presence of protectants, the long-term stability after freeze-drying may still require low temperature storage, in order to inhibit diffusion-dependent destructive chemical reactions. Thus, further innovations have been sought to provide long-term storage of labile biological materials at ambient temperatures.
Storage of dried materials at ambient temperatures would be cost effective when compared to low temperature storage options. Furthermore, ambient temperature storage of biological materials such as vaccines and hormones would be extremely valuable in bringing modem medical treatments to third world countries where refrigeration is often unavailable. As the many benefits of shelf preservation of biological specimens have come to be appreciated, researchers have endeavored to harness vitrification as a means of protecting biological, materials against degradative processes during long-term storage. Consequently, this technology of achieving the xe2x80x9cglassxe2x80x9d state has been anticipated to emerge as a premier preservation technique for the future.
A glass is an amorphous solid state that may be obtained by substantial undercooling of a material that was initially in the liquid state. Diffusion in vitrified materials, or glasses, occurs at extremely low rates (e.g. microns/year). Consequently, chemical or biological changes requiring the interaction of more than one moiety are practically completely inhibited. Glasses normally appear as homogeneous, transparent, brittle solids, which can be ground or milled into a powder. Above a temperature known as the glass transition temperature (Tg), the viscosity drops rapidly and the glass becomes deformable and the material turns into a fluid at even higher temperatures. The optimal benefits of vitrification for long-term storage may be secured only under conditions where Tg is greater than the storage temperature. The Tg is directly dependent on the amount of water present, and may therefore be modified by controlling the level of hydration; the less water, the higher the Tg.
Unfortunately, the advantages of vitrification technology as a means of conferring long-term stability to labile biological materials at ambient temperatures has not been fully utilized. Conventional methods of ambient temperature preservation by desiccation are designed for laboratory processing of very small quantities of materials. Recently, Bronshtein developed an alternative method of preservation by foam formation (U.S. Pat. No. 5,766,520) that is compatible with large-scale commercial operations. Preservation by foam formation overcomes the technical problems related to scaling up desiccation and vitrification preservation processes. For this reason, preservation by foam formation is attractive as a scalable method for long-term storage of biological materials.
The present invention addresses instrumentation problems related to preservation by foam formation and processing operations. Specially designed devices and instruments must be employed to reproducibly produce a dehydrated, shelf-stable, foams and uniform powder of the preserved materials. The instruments may integrate the ability to execute a barrier scalable preservation of biological material by desiccation, subsequent transformation of the dry material into powder form (for example by milling) and usage of dry powders to formulate products that may contain mixtures of different biological materials.
The present invention relates to an industrial scale barrier process for preserving a biologically active material. The process comprises loading an industrial scale volume of a solution or suspension containing the biologically active material into a process vessel adapted to fit within a process chamber. The solution or suspension is subjected to drying conditions, which comprise a temperature and a vacuum pressure, wherein the drying conditions are sufficient to cause the solution or suspension to boil without freezing. The drying conditions are monitored using a temperature sensor and a pressure sensor and are adjusted as required to maintain boiling without freezing by applying heat to the solution or suspension until a mechanically stable foam is formed.
In one embodiment, the biological solution or suspension is combined with a protectant prior to loading into the process vessel. The protectant may be selected from the group consisting of sugars, polyols and polymers. The protectant may further comprise a mixture of a monosaccharide, a disaccharide, an oligosaccharide and a polymer. The monosaccharide may be a non-reducing derivative of a monosaccharide selected from the group consisting of fructose, glucose, sorbose, piscose, ribulose, xylulose, erythulose, and the like. The non-reducing derivative is prepared from monosaccharides having at least one reducing group, wherein the at least one reducing group is modifying by methylation, ethylation, or chlorination.
In one variation of the process, the industrial scale volume is greater than about 0.01 liters. In another variation, the industrial scale volume is at least 10 liters.
The process vessel in accordance with the present invention has a length and a diameter, wherein the ratio of the length to the diameter may be within a range of about 1:1 to about 10:1. The surface to volume ratio of the proceses vessel may be within a range of about 1 to about 25. In one mode, at least a portion of the surface of the process vessel is adapted to facilitate heat transfer between the solution or suspension inside the vessel and a conductive heat source thermally coupled to the vessel surface.
In a variation of the present process, the conductive heat source may comprise a circulating fluid in the temperature range of 5xc2x0 to 100xc2x0 C. In another mode, the conductive heat source may comprise an electrical resistance element. The heat which is applied to the solution or suspension may be generated by one or more of the following: conductive heating, inductive heating, or dielectric heating. In embodiments where inductive heat is applied, a heat transfer surface on the process vessel may be inductively heated by applying a low frequency alternating current of between about 50 to about 500 Hz to a coil of conducting material, wherein the heat transfer surface then conductively heats the solution or suspension in the process vessel. In addition, or in the alternative, the inductive heating may be applied directly to the solution or suspension by applying a high frequency alternating current of between about 5 to about 60 MHz to a coil of conducting material. In embodiments where dielectric heat is applied, the dielectric heating may comprise an electrostatic field generated by applying a high frequency alternating current of between about 5 to about 60 MHz to a capacitor.
In one variation of the process, after the mechanically stable foam is formed, the foam may be subjected to stability drying under vacuum by applying further heat. The further heat applied during stability drying may be generated by inductive or dielectric heating.
In one mode, the process vessel is rotated. In another mode, the process vessel may be a flexible or semi-rigid container. The process vessel may be sealed within the process chamber in accordance with one variation.
In one preferred variation, the process vessel is placed in a cassette prior to loading the solution or suspension, wherein the cassette is adapted to fit within the process chamber. The cassette may be further adapted to thermally couple the process vessel with a heat source.
In accordance with another aspect of the invention, an apparatus is disclosed for performing an industrial scale barrier process of preserving a biologically active material. The apparatus comprises: a process chamber having a vacuum port and a door, which when closed creates a substantially gas-tight interior; a vacuum line connected to both the vacuum port of the process chamber and a vacuum source, the vacuum line further comprising a vacuum sensor adapted to detect a vacuum pressure within the process chamber and generate a corresponding output signal, and a control valve adapted to regulate the vacuum pressure within the process chamber; a process vessel for holding an industrial scale volume of a solution or suspension containing the biologically active material, wherein the process vessel is configured to fit through the door and into the process chamber; a heat source adapted to thermally couple to the solution or suspension in the process vessel; and a temperature sensor adapted to detect a temperature of the solution or suspension and generate a corresponding output signal, wherein the vacuum pressure and temperature are adjusted during the preservation process based on the respective output signals from the pressure sensor and temperature sensor, such that the solution or suspension is caused to boil without freezing until a mechanically stable foam is formed.
In one preferred embodiment, the apparatus may also include a controller in electronic communication with the pressure sensor and the temperature sensor, wherein the controller is also adapted to actuate the vacuum control valve and the heat source, such that the controller monitors the respective output signals from the pressure sensor and temperature sensor and adjusts the vacuum pressure and temperature according to a predetermined relationship, such that the solution or suspension is caused to boil without freezing until a mechanically stable foam is formed.
In one mode, the vacuum source and vacuum control valve are adapted to produce a chamber pressure within a range of about 0 to about 500 Torr. In a further mode, the apparatus also includes a motor for rotating the process vessel. The process vessel in accordance with one aspect of the apparatus may be a deformable container, such as a bag.
The process vessel in accordance with one preferred variation may be configured so as to fit within an outer heating element that substantially surrounds the process vessel, and wherein the process vessel also has a centrally disposed invagination configured to substantially surround an inner heating element, whereby a surface to volume ratio and a resultant capacity to transfer heat is enhanced relative to a configuration without the inner heating element.
The apparatus may include a cassette adapted to receive the process vessel and fit within the process chamber. The cassette may include elements for facilitating heat transfer.
In a variation, the heat source may comprise a conductive heating system, an inductive heating system, a dielectric heating system, or a combination thereof. The conductive heating system may comprise a circulating thermal fluid system, comprising a thermal fluid, a pump, a conduit and a heating element. In addition or in the alternative, the conductive heating system may comprise an electrical resistance element thermally coupled to the process vessel. The inductive heating comprises a coil of conducting material coupled to a power source adapted to generate an alternating current of between about 5 MHz to about 60 MHz. The dielectric heating system comprises a capacitor coupled to a power source adapted to generate an alternating current of between about 50 Hz to about 60 MHz.